Mechanism of Virulence Regulation by Membrane Activators

描述(申请人提供)：霍乱弧菌的毒力基因调控需要四种不寻常的转录调节蛋白的作用，即称为ToxR/ToxS和TCPP/TcpH的膜定位转录激活/效应分子对。这些蛋白质作用机制的工作模型认为，ToxR和TCPP合作激活一种蛋白质ToxT的表达，ToxT激活编码致病因子霍乱毒素的基因和毒素共同调节的菌毛。相比之下，ToxR独立于TCPP，可以调节两种外膜蛋白OmpU和OmpT的表达。激活剂和效应器之间的相互作用预计发生在周质空间，尽管一般而言，效应器的作用还不太清楚。TcpH作用于阻断针对TCPP周质结构域的蛋白降解机制。本研究将评估TcpH是否在TCPP的功能中发挥其他作用。FOX可能为ToxR赋予其功能所必需的高阶结构。从这些膜局部激活器功能的机制模型中产生的特定假设将被检验。我们对这些蛋白质的大部分了解来自于遗传和生化研究，这些研究将在这里提出的工作中继续下去。随着原核细胞研究的进展和成像和细胞学资源的提供，还提出了研究转录复合体的膜定位的试剂和实验方法。 具体目标 确定ToxR激活ompU和toxT的机制。 确定TCPP的DNA结合和RNA聚合酶相互作用在毒素T激活中的作用。 生病了。确定激活剂/效应器周质相互作用的机制和后果。 建立细胞学方法分析膜定位激活蛋白的功能。

DESCRIPTION (provided by applicant): Virulence gene regulation in Vibrio cholerae requires the action of four unusual transcription regulatory proteins, pairs of membrane localized transcription activator/effector molecules called ToxR/ToxS and TcpP/TcpH. Working models for the mechanisms of action of these proteins hold that ToxR and TcpP collaborate to activate expression of a protein ToxT, which activates genes encoding virulence factors cholera toxin and toxin co-regulated pilus. By contrast, ToxR alone - independently of TcpP - can regulate expression of OmpU and OmpT, two outer membrane proteins. Interactions between the activators and effectors are predicted to take place in the periplasmic space, although in general the roles of the effectors are less well characterized. TcpH acts to block a proteolytic mechanism that targets periplasmic domain of the TcpP. Whether TcpH plays any other role in the function of TcpP will be assessed in this study. FoxS may serve to confer higher order structure on ToxR essential for its function. Specific hypotheses generated from these mechanistic models of membrane-localized activator function will be tested. Much of what we understand about these proteins has come from genetic and biochemical studies, and these will continue in the work proposed herein. With advances and imaging and cytological resources available for studying the prokaryotic cell, studies aimed at developing reagents and experimental approaches for studying membrane localization of transcription complexes are also proposed. Specific Aims I. Determine the mechanism of ompU and toxT activation by ToxR. II. Determine the role of DNA binding and RNA polymerase interaction by TcpP for toxT activation. Ill. Define the mechanisms and consequences of activator/effector periplasmic interactions. IV. Develop cytological methods for analyzing membrane localized activator function.